Ultra-fast detection and differentiation of Brucella genus bacteria, B. abortus, B. melitensis, and B. suis, using multiplex convection polymerase chain reaction

Brucellosis is a global zoonotic disease caused by facultative intracellular bacteria from the genus Brucella. Brucella spp. are gram-negative bacteria that are pathogenic to humans and a variety of animals. Rapid detection and timely treatment of brucellosis is important for increasing the curative rate, to prevent spread among animals, and to reduce the risk of transmission to humans. In this study, we developed a rapid multiplex convection polymerase chain reaction (cPCR) to detect and differentiate B. abortus, B. melitensis, and B. suis. In the ultra-fast cPCR method, a universal primer IS711 and species-specific primer sets specifically detected each target species after a 24 min amplification reaction. Multiplex detection was performed with a mixture of the universal primer and all species-specific primers. Species-specific DNA amplicons were clearly identified by their expected sizes within the same amplification time. When sensitivity of detection was tested, approximately 28 genome equivalents (0.1 pg of genomic DNA) were detected using the cPCR system. The cPCR operation time could be reduced to 20 min, 25 cPCR cycle, without losing the sensitivity of detection. The assay developed in this study worked well in the presence of corresponding animal genomic DNA. The results revealed that Brucella species can be detected and differentiated in singleplex and multiplex regimes of ultra-fast cPCR. The ultra-fast speed and the sensitivity of molecular detection and differentiation of Brucella species developed in this study may be useful in laboratory and field applications.